Characterization of proteins often requires the ability to incorporate detectable groups—e.g., fluorochromes, chromophores, spin labels, radioisotopes, paramagnetic atoms, heavy atoms, haptens, crosslinking agents, and cleavage agents—at specific, defined sites. For proteins that do not contain pre-existing cysteine residues, site-specific labeling can be accomplished by use of site-directed mutagenesis to introduce a cysteine residue at the site of interest, followed by cysteine-specific chemical modification to incorporate the labeled probe. However, for proteins that contain pre-existing cysteine residues, site-specific labeling is difficult. Multiple strategies have been reported: (i) intein-mediated labeling (“expressed protein ligation”), (Muir, et al., Proc. Nat'l. Acad. Sci. USA, 95:6705-6710 (1998)); (ii) transglutaminase-mediated labeling (Sato et al., Biochem. 35:13072-13080 (1996)); (iii) oxidation-mediated labeling (Geoghegan, et al., Bioconj. Chem., 3:138-146 (1992)); (iv) transition-metal-chelate-mediated labeling (Hapanidis et al., J. Amer. Chem. Soc., 123:12123 (2001)) and (v) trivalent-arsenic-mediated labeling (Griffin et al., Science 281:269-272, 1998) (U.S. Pat. No. 6,008,378). Strategies (i)-(iii) do not permit in situ labeling (i.e., direct labeling of proteins in cuvettes, gels, blots, or biological samples—without the need for a subsequent purification step) or in vivo labeling (i.e., direct labeling of proteins in cells). Strategy (iv) does not permit labeling and analysis at subnanomolar concentrations. Strategy (iv) requires a structural scaffold presenting two trivalent-arsenic atoms in a precisely defined spatial relationship and therefore relates only to a limited number of detectable groups (such as those having an isoxanthenone/resorufin structural nucleus).
The ability to observe proteins in living systems in cells or in situ can provide important information on expression and activity of the proteins. This information is useful in such wide applications as molecular biology research to histological diagnostics and identification of useful drug candidates. It is well known to use dyes of various types to impart a detectable property to a target material. For example, the technique of bonding a detectable compound, such as a fluorescent dye, to a substituent which is reactive with the target material is used to render the target compound detectable by methods such as fluorescence microscopy, fluorescence immunology and flow cytometry.
Various fluorescent dyes are known, including those based on fluorescein (green fluorescence), rhodamine (orange fluorescence), and coumarin and pyrene chromophores (blue fluorescence). However, use of these dyes may at times be problematic. For example, dyes based on fluorescein have a tendency to photobleach when illuminated with strong excitation sources. The rapid loss of image over time can interfere with detection and quantification of targets labeled using these dyes. Furthermore, fluorescein derivatives have a pH sensitive absorption spectrum with a marked decrease in signal below a pH of 8. Rhodamine derivatives are hydrophobic and are difficult to use in aqueous media. They often show strong fluorescence quenching when bound to proteins. Molecular Probes—Handbook of Fluorescent Probes and Research Chemicals, Haugland, R. Ed., (1996). Cyanine dyes possess superior photostability as compared to fluoroscein, better water solubility than the rhodamine dyes, and are stable in the range of pH 3 to 10. For this reason, cyanine dyes have been found particularly useful in labeling biological targets.
When the target material is a biological compound, in cells or in situ, a number of challenges are introduced which limits the utility of many of the known fluorescent dyes. In these applications, in order for a particular dye to be effective, it must first cross the cell membrane. As a result, large dyes, such as phycobiliproteins (molecular weights of from 33,000 to 240,000) are not useful because they are unable to enter the cell to bind with the target material. In addition, when the target is a metabolite, a drug, a hormone, or the like, the dye may be so large that it either interferes with the activity of the target or possesses steric limitations which prohibit it from binding to the target at all.
A further challenge when detecting biological compounds in situ is the need for specificity of association with the target material. Since excess probe cannot be “rinsed off” in these applications, unbound or non-specifically bound probe will introduce noise into the system. Some dyes are known to have a certain degree of specificity for certain endogenous reactive groups or tags in the target material. For example, U.S. Pat. No. 6,225,050 B1 to Waggoner discloses use of certain luminescent cyanine dyes containing a group which is covalently reactive with amine or hydroxyl groups and is used to label biological compounds possessing these groups such as proteins, nucleic acids, cells, sugars, and the like. The fact that amine and hydroxyl groups are numerous and widely dispersed on a protein target is used to facilitate detection of the fluorescent probe or luminescent cyanine dye because it becomes attached at multiple sites on the protein therefore generating a strong signal.
The method of Waggoner may be used to quantify a biological compound such as a protein, if the number of reactive sites thereon is known, by dividing the total luminescence intensity by the number of reactive groups to determine the total amount of protein. However, if the sample contains any other biological compounds having the reactive amine or hydroxyl groups, then the probe will bind to these compounds as well. Thus, in a mixed sample, the measured luminescence will not reflect the concentration of the target but rather will reflect the presence of any and all compounds having these groups. Since most biological compounds do contain these groups, the method is not useful in detecting the presence of a protein in a mixed sample, in a cell, or in situ. In some applications it is possible to first separate the proteins from the rest of the sample and then detect luminescence. However, this requires an additional step, and removes the protein from the sample. As a result, this method is impractical for measurement of protein in situ, protein-protein interactions, and the like.
The Waggoner patent also discloses a two step labeling procedure. First, a primary component such as an antibody is labeled with the dye. Next, the labeled antibody is used as a probe for the secondary component, such as an antigen for which the antibody is specific. Monoclonal antibodies, which bind with specificity to cell surfaces and the like, are particularly useful in this regard. In fact, fluorescein, Texas Red, rhodamine and phycoerythrin labeled monoclonal antibodies are now commercially available. However, application of this method is limited to those secondary components or target materials which bind specifically to a particular monoclonal antibody. Thus, these dyes can only be specific to certain compounds and are therefore limited in their application.
Rather than rely on endogenous reactive groups or antigens as binding sites, it is known to engineer a small receptor motif into the protein as a tag. A dye or probe can then selectively bind to these motifs or targets. Intein mediated labeling or expressed protein ligation and oxidation mediated labeling methods are known methods for labeling receptor motifs at protein termini. However, these methods cannot be used to label internal sites within a protein. In addition, these methods suffer the same drawbacks as the Waggoner probes, as they require removal of excess probe for accurate detection of proteins. As a result, these methods also fail to achieve direct labeling of proteins in cuvettes, gels, blots or biological samples without a further purification step. Furthermore, these probes are unsuitable for in situ detection schemes.
U.S. Pat. No. 6,008,378 to Tsien et al. discloses a bis-arsenical compound useful in detecting the presence of a biological target material. The bis-arsenical compound (known as FlAsH) is used to label proteins by reacting with a target sequence of a thiol containing tetracysteine motif (Cys4). FlAsH may be modified to contain a detectable group such as a fluorescent group. The FlAsH label may be further modified to include dithiol groups for protection against binding to low affinity sites, such as endogenous cysteine residues or dihydrolipoic acid moieties. A preferred dithiol is 1,2-ethanedithiol (EDT). The FlAsH-EDT complex can be non-fluorescent when it is not bound to a target sequence, which can aid in reduction of background noise caused by unbound FlAsH.
The FlAsH compound reacts with a certain amount of specificity to a Cys4 motif target sequence that has been incorporated into the target material. Specifically, the thiol groups of the cysteines in the target sequence react with the arsenical moieties of FlAsH. However, there is significant noise in the system. Although an improvement over other dyes in that unbound probe can avoid contributing to background noise when it is non-fluorescent, the FlAsH-EDT complex has been found to have less specificity to the Cys4 motif than originally thought. In one study, FlAsH-EDT was found to fluoresce significantly when added to protein homogenates from non-transfected cells that lacked the Cys4 motif. Stroffekova, K., European J. of Physio., 442:859-866 (2001). Thus, this system produces background noise whereby the biarsenical compound binds to other sulfur molecules, such as those contained in other cysteine or lysine amino acids, besides those in the target sequence.
In protein analysis, it is often important to not only detect the presence of a protein, but also to determine spatial relationships within a protein or to detect protein-protein interactions.
Fluorescence resonance energy transfer (FRET) is a physical phenomenon that permits measurement of molecular distances. FRET occurs in a system having a fluorescent probe serving as a donor and a second fluorescent probe serving as an acceptor, where the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. In such a system, upon excitation of the donor with light of the donor excitation wavelength, energy can be transferred from the donor to the acceptor, resulting in excitation of the acceptor and emission at the acceptor emission wavelength. With fluorochromes and chromophores known in the art, FRET is useful over distances of about 1 nm to about 15 nm, which are comparable to the dimensions of biological macromolecules and macromolecule complexes. Thus, FRET is a useful technique for investigating a variety of biological phenomena that produce changes in molecular proximity. When FRET is used as a detection mechanism, colocalization of proteins and other molecules can be imaged with spatial resolution beyond the limits of conventional optical microscopy.
Among the most widespread donor acceptor pairs are luminescent cyanine dyes including Cy3/Cy5, and Cy5/Cy7. The pairs typically include a target bonding group capable of forming a covalent bond with a target sequence or target compound.
U.S. Pat. No. 6,130,094 to Waggoner discloses a FRET method using pairs of fluorescent dyes for use as probes which bind to cellular constituents. Low molecular weight fluorescent labeling complexes are used having large wavelength shifts between absorption of one dye and emission from another dye in the complex. The dyes are attached through linkers to form donor-acceptor complexes. FRET from an excited donor to a fluorescent acceptor provide the detectable signal. The dyes contain reactive groups for labeling functional groups on target compounds. A disadvantage of this invention is that the reactive group may not be specific to just the functional group on the target compound. Furthermore, the dyes must first be pre-assembled using the linker before they are added to the sample, and therefore will emit a detectable signal even if not bound to a target material. As a result, less than complete bonding of each complex to targets may generate false positive signals or background noise unless unbound complex is first removed from the sample. This is particularly problematic when attempting to identify small amounts of protein, as is typically the case when observing proteins in living cells. As in the previously described probes, this method is impractical for detection of proteins in cells, in situ or for any direct reading applications.
There is a present need for detectable probes capable of selectively binding to biological target materials in cells, in situ or other indirect reading applications. Accordingly, a need exists for a fluorescent probe which exhibits distinguishable fluorescent characteristics when attached to a target material as opposed to when unattached to target material. A further need exists for a fluorescent probe in which background noise, generally, and non-selective binding to unintended sites, in particular, is reduced. These and further objectives are provided by the methods and probes of the present invention.